Evaluation of the effects of ethanol and mitomycin on survival of rat limbal stem cells: an in vitro study.

Abstract

Ethanol and mitomycin C (MMC) are clinically used to treat corneal diseases such as LASEK and LASIK surgery. In this study, we investigated the effects of time-dependent alcohol and MMC in cultured rat limbal stem cells (LSCs) to determine the appropriate time for the use of this compound in the clinical setting. LSCs (N = 10 eyes) isolated from male Wistar rats were cultured and characterized; then, isolates were divided into three groups. One group was exposed to a 20% concentration of ethanol for 5, 10, 15, 20, 25, and 30s, and cell viability was assessed one, three, and five days following ethanol exposure using an MTT assay. To investigate the effect of MMC, cells in the second group were treated with 0.02% MMC in various periods (i.e., 15s, 30s, 60s, 90s, and 120s) and time-dependent responses of cultured LSCs were recorded. Cells in the third group were co-treated with ethanol and MMC; then, dose and time dependency was evaluated. In comparison with the viable cells in the control group, ethanol markedly decreased the viability of cells in a time-dependent manner in days one and three. On day five, the viability of LSCs was improved significantly (p < 0.05) in comparison with day one. The number of viable progenitor cells was significantly decreased after MMC treatment in a time-dependent manner, as determined by the MTT assay (p < 0.001). The use of mitomycin, along with alcohol, decreased cell viability in all groups treated with ethanol + MMC compared to the control on days one, three, and five (p < 0.0001). Our findings suggest that ethanol and MMC reduced cell viability in cultured LSCs in a time-dependent manner. In addition, when LSCs were exposed to alcohol alone, they had a better recovery process within 5days in comparison to when exposed to mitomycin alone or mitomycin + alcohol.